Glioblastoma multiforme (GBM) is almost universally fatal. The discovery of tumor-initiating cells with the capacity to self-renew, sometimes termed cancer stem cells, has created tremendous enthusiasm for the development of new avenues of therapy. These cells utilize familiar pathways for their proliferation, such as the PI3 Kinase pathway. Despite the hope raised by the discovery of brain tumor stem cell-like cells, numerous obstacles lie in the path of therapeutic development. One complication is that these cells have significant resistance to conventional therapies and to inhibition of pathways. Another is that there are differences amongst brain tumor stem-like cells that are present in the tumors of different patients. The goals of this study are to critically examine brain tumor stem cell-like cell biology in order to develop the means to attack them and to overcome their mechanisms of resistance. First, we will examine the heterogeneity of GBM stem cell-like cells through the use of recent advances by the The Cancer Genome Atlas (TCGA). We will obtain samples from patients and group them according to molecular subclasses defined through the analysis of gene expression. We will evaluate the ability of these cells to give rise to neurospheres in vitro as well as to form tumors in xenografts. We will then use a pharmacologic and gene manipulation strategy to determine the dependence of GBM stem cell-like cells on different nodes of the PI3K pathway. We will determine whether the four subgroups defined by the TCGA--Neural, Proneural, Mesenchymal and Classical--confer different levels of dependency on these nodes for proliferation and tumorigenesis. We will next assess the role of the PI3K pathway in mediating the enhanced resistance to radiation observed in brain tumor stem cell-like cells. We will test the hypothesis that activation of the pathway results in enhanced resistance to radiation in vitro and determine whether we can reverse this resistance through inhibition of specific pathway components. Then we will test the hypothesis that one of the mechanisms by which pathway activation promotes radiation resistance is through the activation of the Nrf2 oxidative stress-response mechanism. We will then explore mechanisms of chemoresistance in GBM stem cell-like cells. We will use cell culture, in vivo assays and a new microfluidicsbased immunocytochemical analysis (MIC) system to determine whether rapamycin selects for stem cell-like cells with enhanced tumorigenicity and pathway activation. We will also determine whether resistance to rapamycin treatment can be overcome through inhibition of hyperactivated pathways. Then, we will identify novel pathways of resistance based on a completed phosphoproteomic screen to discover proteins that are phosphorylated or dephosphorylated during the development of rapamycin resistance. We will determine the potential role of the proteins identified by this screen in the development of resistance. These collaborative studies will pave the way for a deeper understanding of GBM biology and inform future clinical and translational and clinical research into the mechanisms and treatment of GBM.